Review Article - Hindawi Publishing...

Hindawi Publishing CorporationJournal of Biomedicine and BiotechnologyVolume 2011, Article ID 981214, 12 pagesdoi:10.1155/2011/981214

Review Article

The Gut Microbiota and Human Health with an Emphasis onthe Use of Microencapsulated Bacterial Cells

Satya Prakash, Catherine Tomaro-Duchesneau, Shyamali Saha, and Arielle Cantor

Biomedical Technology and Cell Therapy Research Laboratory, Departments of Biomedical Engineering and Physiologyand Artificial Cells and Organs Research Center, Faculty of Medicine, McGill University, 3775 University Street, Montreal,QC, Canada H3A 2B4

Correspondence should be addressed to Satya Prakash, [email protected]

Received 19 November 2010; Revised 16 February 2011; Accepted 11 April 2011

Academic Editor: Eric C. Martens

Copyright © 2011 Satya Prakash et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The gut microbiota plays a crucial role in maintaining health. Alterations of the gut bacterial population have been associated with anumber of diseases. Past and recent studies suggest that one can positively modify the contents of the gut microbiota by introducingprebiotics, probiotics, synbiotics, and other therapeutics. This paper focuses on probiotic modulation of the gut microbiota bytheir delivery to the lower gastrointestinal tract (GIT). There are numerous obstacles to overcome before microorganisms can beutilized as therapeutics. One important limitation is the delivery of viable cells to the lower GIT without a significant loss of cellviability and metabolic features through the harsh conditions of the upper GIT. Microencapsulation has been shown to overcomethis, with various types of microcapsules available for resolving this limitation. This paper discusses the gut microbiota and its rolein disease, with a focus on microencapsulated probiotics and their potentials and limitations.

1. Introduction

The gut microbiota, which resides in the gastrointestinal tract(GIT) and is also termed microflora, plays an importantrole in human health and disease. The GIT is comprised ofthe stomach, the duodenum, the jejunum, the ileum, thecolon, the rectum, and the anal canal. The lower digestivetract, specifically the colon, is the primary site of importancefor bacterial cell colonization; however, upper digestivetract microorganisms are also of importance. The bacterialpopulation of the gut has been studied in diseases such ascolon cancer, inflammatory bowel diseases (IBD), hyper-cholesterolemia, nonalcoholic fatty liver disease (NAFLD)and others. Certain bacterial populations, such as lactic acidbacteria, have been shown to positively influence health.Hence, attempts to modify the microflora, towards thosebacteria, for disease treatment and prevention should proveadvantageous. For this purpose, prebiotics, probiotics, andsynbiotics have been used. The delivery of viable probioticbacteria is impeded by the harsh conditions of the upperGIT, hence, a vessel for delivering optimum cell viability tothe lower GIT is required. Microcapsules can be used as a

vehicle with the capability to protect the viability and activityof orally delivered bacterial cells through the upper GIT.

This paper will first give an overview of the gut micro-biota and its main characteristics, focusing on its role incolon cancer, IBD, and hypercholesterolemia. Modulation ofthe gut microbiota to promote health will then be describedthrough the use of probiotics, prebiotics, and synbiotics, withprobiotics as a main focus. Microencapsulation and types ofmicrocapsules will be described along with their success inthe treatment and prevention of diseases. Finally, the paperwill conclude with a discussion on this field’s future.

2. The Gastrointestinal Bacterial System

The gut microbiota contains a broad spectrum of microor-ganisms, totalling 1013 to 1014 bacterial cells, but has notbeen completely explored as of yet [1]. The importance ofthe gut microflora is exemplified by the fact that the numberof bacterial cells outnumbers human cells by a factor often [2]. The human intestinal habitat contains 300 to 500different species of bacteria, varying significantly in content

2 Journal of Biomedicine and Biotechnology

AnatomicalGIT region

pH ofGIT

region

Main contents of the region

Prominentbacterial

populations

StomachPepsin, amylase(from salivary

glands), mucus

Lactobacilli,streptococci

Smallintestine

DuodenumJejunumIleum

Largeintestine

Bicarbonate, mucus

Aerobic organisms(upper GIT)

Anaerobic organisms

(lower GIT)

Number of bacterial

cells/gram of GIT region contents

Pancreaticenzymes,

bicarbonate, bilesalts, mucus

Ascending colonTransverse colonDescending colon

Lactobacilli,Escherichia coli,

EnterococcusFaecalis

Lactic acid bacteria,Bacteroides,

Bifidobacteriumbifidum

< 103

104−7

1010−12

1-2

6-7

5-7

Figure 1: The GIT characteristics (oxygen distribution, pH, bacterial populations, and bacterial cell counts) and the localization of thevarious gut bacterial populations, termed microbiota.

between individuals [3]. Most gut bacteria reside in the lowerpart of the digestive tract, in the large intestine, since theupper tract consists of high levels of acid, bile, and pancreaticsecretions which are toxic to most microorganisms, as shownin Figure 1 [3]. Even though some bacterial species of thegut are potential pathogens, the constant interaction betweenthe host and its microbes usually remains beneficial tothe health of the host [4]. It has been demonstrated thatthe gut bacterial population plays an important role intheir host’s metabolism and energy consumption, especiallyin the digestion and absorption of nutrients [1, 5]. Theupper portion of the GIT, made up of the stomach andthe duodenum, harbours very low numbers of microor-ganisms, with less than 1000 bacterial cells per gram ofcontents, with the predominant microorganisms presentbeing Lactobacilli and Streptococci [6, 7]. The relatively lownumber of microorganisms found in the upper digestivetract, although some are of great importance in humandisease, can be explained by the presence of high levelsof acid, bile, and pancreatic secretions, as aforementioned[7, 8]. One important organism found in the stomach, whichcan withstand these harsh conditions, is Helicobacter pylori,a microorganism responsible for ulcers and stomach cancer[9]. There is also a phasic propulsive motor activity in the

upper GIT which impedes any stable bacterial colonization[10]. Lower in the digestive tract are found the jejunum andthe ileum where there is a gradual increase in the bacterialnumbers from 104 to approximately 107 cells per gram ofcontents by the time the distal ileum is reached [7]. Once inthe large intestine, the tract is heavily populated by anaerobeswith up to 1012 cells per gram of luminal contents [10]. Thispaper focuses on the lower part of the digestive tract, due toits abundant bacterial population.

Bacteria are classified into genera and species based ontheir individual phenotypic and genotypic characteristics,with a number of different genera found actively residingin the human GIT. The dominant anaerobic genera areBacteroides, Bifidobacterium, Eubacterium, Clostridium, Pep-tococcus, Peptostreptococcus, and Ruminococcus [4, 11]. Themain genera of facultative anaerobic bacteria are Escherichia,Enterobacter, Enterococcus, Klebsiella, Lactobacillus, and Pro-teus [4, 11]. The proportion and numbers of these bacteriacan vary, depending on a number of genetic and envi-ronmental factors, including disease state and one’s foodintake [1, 11–13]. The main functions of the microflora weremostly elucidated by investigations with animals bred undergerm-free conditions, with the functions broadly qualified asmetabolic, trophic, and protective [3, 14, 15].

Journal of Biomedicine and Biotechnology 3

The gut microbiota has a significant impact on host me-tabolism, participating in microbial-mammalian co-metab-olism. The microbiota is considered a multifunctional organwith metabolic capabilities that humans have not yet fullyevolved into their own genomes [16]. It has the abilityto break down indigestible plant polysaccharides, termeddietary fibers and also plays an important role in thebiotransformation of conjugated bile acids, described inmore detail later in this paper [17–20]. The importance ofthe gut microbiota in vitamin synthesis was demonstratedmany years ago with the use of germ-free animals [21].Experiments on a chick animal model demonstrated thesynthesis of riboflavin, vitamin B, pantothenic acid, vitaminB12, folic acid, nicotinic acid, thiamine, and biotin by the gutmicrobiota [22]. Furthermore, Pseudomonas and Klebsiellasp., two resident organisms of the small intestine, were specif-ically shown to synthesize significant amounts of vitamin B12

[23]. As described, the gut microbiota has extensive roles toplay in normal human metabolism.

3. The Gut Microbiota and Its Role inHuman Health and Disease

The gut microbiota has gained importance in disease aetiol-ogy and pathology, with emerging evidence demonstratingits role in disease [1, 24]. A number of diseases have beenassociated with alterations of the gut microbiota, and if onecan elucidate the exact link between the two one can beginto successfully treat and prevent these disorders through themodulation of the number and/or species of microorganismspresent. Some disorders associated with the microflorainclude colon cancer, IBD, hypercholesterolemia and nonal-coholic fatty liver disease, among others [13, 24–28].

3.1. Role of the Gut Microbiota in Colon Cancer. Colorectalcancer is the second most common cause of cancer deathin men and women [29]. Although the genetic mechanismsof colorectal cancer are well established, there are severalenvironmental factors that have also been implicated in thedevelopment of sporadic colon carcinomas [3, 30]. Foods,such as processed meats, which contain high levels of dietaryfat, have been associated with an increased risk of coloncancer development when compared to the risk associatedwith a high intake of fruits, vegetables, grains, and fish [30,31]. It was proposed that the effect of diet could be mediatedby changes in the composition of the colonic microflorasuch that the intestinal bacteria are responsible for theinitiation of colon cancer [3, 13]. Data shows that bacteriaof the Bacteroides and Clostridium genera were associatedwith an increase in the incidence and growth rate of colonictumors in tumor-induced animals, while genera such as Lac-tobacillus and Bifidobacterium (well-characterized bacteriapredominantly used in therapeutic probiotic formulations)appeared to prevent tumorigenesis [32, 33]. The propertiesof the colonic microflora make it a promising target for thedevelopment of a colon cancer therapeutic [34].

3.2. Inflammatory Bowel Disease and the Gut Microbiota.IBD, prominent in Western countries, is made up of a group

of disorders that are characterized by a chronic and relapsinginflammation of the GIT [35]. The two most prominentforms of IBD are Crohn’s Disease (CD) and ulcerative colitis(UC), with the bacterial flora as an important factor andcontributor of the inflammation [36, 37]. Sufferers of IBDhave a higher bacterial attachment to gut epithelial surfaceswhen compared to that found in healthy individuals [38].The link between intestinal mucosal inflammation and theresident bacteria has been further demonstrated, in vivo,using rats and mice treated with broad-spectrum antibiotics[39]. This treatment mitigates, although only temporarily,mucosal inflammation in animals with IBD, suggesting thatthe resident bacteria are causing the inflammation [39].Furthermore, an overpopulation of the Bacteroides generaon the gut epithelium leads to an increased occurrence oftransmural inflammatory lesions [3]. Early research demon-strated that the presence of Escherichia coli is linked to activeUC and contributes to the development of inflammation[40]. E. coli has also been linked to CD, with the presence ofspecific adherent-invasive species found in the resected ileumof patients [41, 42]. This effect appears to be species specificas only certain phylogenetic groups of E. coli were found to bemore frequent in UC and CD patients when compared withhealthy controls [43, 44]. It is clear that the gut microfloraplays an important role in IBD pathology and an efficienttherapy is still required.

3.3. Gut Microbial System and Hypercholesterolemia. Hyper-cholesterolemia is a disorder whereby an individual demon-strates an elevated serum cholesterol level. For many decadesnow this disorder has been recognized as a significant riskfactor associated with atherosclerosis and coronary heartdisease [45]. Current treatment options to lower serumcholesterol levels involve the use of pharmacological agentssuch as statins which act by inhibiting HMG-CoA reductase,the rate-limiting enzyme of cholesterol biosynthesis [46].Statins make up a group of compounds that are generallywell tolerated but remain expensive and have significantside-effects, including gastrointestinal problems, such asdiarrhoea, but may also include severe liver and skeletalabnormalities [47–49]. Bearing the potential significant con-sequences of hypercholesterolemia in mind, the importanceof the gut microbiota in cholesterol metabolism and thepathogenesis of hypercholesterolemia, a new paradigm issuggested for the development of a successful treatment.

As early as 1959, research was performed to elucidate therole of the gut microbiota in cholesterol homeostasis withresearchers demonstrating that germ-free rats, administereda diet without significant amounts of cholesterol, nonethelessshowed higher serum-cholesterol values than control ratsadministered the same diet [50]. Several mechanisms havebeen proposed as methods by which the gut microbiotamay modulate cholesterol levels within the host [51]. Recentdevelopments have demonstrated that the composition ofthe microbiota and diet is directly correlated with cholesterollevels in vivo, specifically, the number of Bifidobacteriafound in the gut is positively correlated with higher levelsof high-density lipoprotein (HDL) [52–54]. In contrast,the number of Coriobacteriaceae is correlated with higher


levels of non-HDL cholesterol [53]. Gut microbial activitiesinfluence lipid metabolism, bearing a significant impacton hypercholesterolemia, by the modification of bile acidmetabolic patterns, by impacting the emulsification, absorp-tion, and storage properties of bile acids and by influencingthe lipoperoxidation through bile acid signalling properties[19]. With these facts in mind, the modulation of the gutmicrobiota could potentially decrease hypercholesterolemiain affected patients.

4. Modulation of the Gut Microbiota forHuman Health Benefits

Past and current research has demonstrated that the gutmicrobiota plays an important role in the pathogenesis ofa number of diseases. Certain bacteria, considered “good,”such as Bifidobacteria and Lactobacilli, are shown to becorrelated with a decrease in the occurrence of a numberof disorders, suggesting that the targeted increase of thesebeneficial bacteria could decrease the incidence and severityof prominent diseases. The colonic delivery of prebioticsand probiotics are methods that have been successfullyused to modify the gut microbiota. Antibiotics can provebeneficial in short-term use but their prolonged use mayresult in significant side-effects. An important concernis the development of bacterial resistance which reducesthe effectiveness of the therapy and further predisposesthe patient to life-threatening illnesses caused by potentialpathogens with increased resistance to the antibiotic.

Current research focuses on prebiotics, probiotics, and acombination of both, termed synbiotics for modulating thegut microbiota. The Food and Agriculture Organization ofthe United Nations defines a prebiotic as a “non-viable foodcomponent that confers a health benefit on the host, asso-ciated with a modulation of the microbiota” [55]. Prebioticmolecules consist of naturally occurring or synthetic sugarsused by certain colonic bacteria, especially Bifidobacteria, asa carbon source for growth and metabolism [56]. Numerousprebiotics have demonstrated their beneficial effects ondisease through modulators of the gut microbiota [57–61].Prebiotic delivery nonspecifically increases the number of“good bacteria” not acting at the species level, which maybe important in some disease states. On the other hand,probiotics are a method by which the gut microbiota canbe specifically modulated for an individual to reestablish andmaintain a healthy state.

4.1. Modulation of the Gut Microbiota by Probiotics Can Pro-mote Human Health. The FAO and WHO define probioticsas “live microorganisms which, when administered in ade-quate amounts, confer a health benefit on the host” [62].Probiotics are inexpensive, safe, free of long-term nega-tive side-effects, and have already demonstrated beneficialeffects for treating immunological, digestive, and respiratorydiseases [62]. Furthermore, these are naturally occurringorganisms found in foods such as milk and yoghurt and,so, are widely accepted by the general public. The mostcommon types of probiotic microorganisms are the lactic

acid bacteria, important components of the healthy gutmicrobiota and regarded as safe by the American FDA [63].Other microorganisms occasionally used as probiotics areyeasts and filamentous fungi [63]. In this section, we describethe use of probiotics on colorectal cancer, IBD, hypercho-lesterolemia and NAFLD.

Probiotics have been proposed and investigated as apotential treatment/prevention method for colorectal can-cer. Early studies demonstrated that 1,2-dimethylhydrazine-(DMH-) induced colon cancer in rats showed a decreasein mortality rate if the test animals were fed Streptococcusthermophilus-fermented skim milk [94]. Another researchgroup demonstrated that Lactobacillus rhamnosus GG, inlyophilized form incorporated in a high-fat diet, was effectiveat reducing tumor incidence in the rat DMH colon cancermodel [95]. Studies using Bifidobacterium longum alsodemonstrated an inhibition of carcinogen-induced coloncancers and precursor lesions [96, 97]. Additional studiesdemonstrate a reduction of colon tumorigenesis markersfollowing the incorporation of Lactobacillus acidophilus ina high-fat control diet in DMH colon cancer rats [98]. Theprobiotics are suggested to achieve a protective effect byinteracting with the carcinogen(s) in the intestinal lumen (inthe case of the DMH rat model, interaction with the DMHmetabolites azoxymethane or methylazoxymethane) leadingto a decrease in the potency/availability of the carcinogeniccompound [97, 98].

Probiotics have also been investigated as a method oftreatment for IBD. A trial in ulcerative colitis (UC) patientswas performed to study the effect of the delivery of an oralprobiotic capsule on the remission of the disorder [99]. Theprobiotic Bifidobacteria were administered following treat-ment with an UC standard therapy [99]. It was demonstratedthat 93.3% of the patients in the control group suffereda disease relapse compared to only 20% of the patientsadministered the probiotic capsule [99]. A significant reduc-tion in inflammation was also observed in the treatmentgroup when compared to the control group [99]. Anotherstudy demonstrated the use of Faecalibacterium prausnitziias a probiotic for treating Crohn’s Disease (CD) [36]. F.prausnitizii and its supernatant were both found to haveanti-inflammatory effects in vitro using peripheral bloodmononuclear and colon adenocarcinoma cells and in vivo ina mouse model of induced colitis [36]. A number of otherstudies related to the effects of probiotics on the preventionand the treatment of IBD, described in another review, havebeen done, with varying success [100].

Early studies suggest that probiotic bacteria may havea beneficial effect on hypercholesterolemic patients, bydecreasing blood lipid levels [101]. A study was undertakenwith hypercholesterolemic mice administered low levels ofthe probiotic Lactobacillus reuteri for a week [101]. Themice demonstrated a decrease in cholesterol and triglyceridelevels and an increase in the HDL : LDL ratio [101]. Astudy was also performed with hyperlipidemic patients whowere administered the probiotic Lactobacillus sporogenes overa three-month period [102]. Following treatment, thesepatients showed, on average, a 32% reduction in totalcholesterol levels accompanied with a 35% reduction in


(2) Blocking ofpathogenic bacterialcells adhesion sites

(1) Production ofpathogen inhibitory

substances

Gut lumen

Gut epithelium

Mucous layer

Bloodstream

Probiotic bacteria

Potentially pathogenic bacteria

Inhibitory substances

Nutrients

Toxin receptors

Toxins

Antibodies

Antigen presenting cells

(3) Nutrientcompetition and

production

(4) Degradation oftoxins and toxin

receptors

(5) Modulation of theimmune responses

Figure 2: Pathways by which a probiotic can positively influence human health. They can influence human health by (1) production ofpathogen inhibitory substances; (2) blocking of pathogenic bacteria adhesion sites; (3) nutrient competition and production; (4) degradationof toxins and toxin receptors; (5) modulation of innate immune responses.

LDL [102]. Studies have also demonstrated that the deliveryof certain strains of Lactobacilli can alleviate symptomsassociated with IBD [103, 104].

Probiotics have also been proposed as a potential treat-ment option for NAFLD because of their modulating effecton the gut flora that could influence the gut-liver axis towardsa healthy state. NAFLD is characterized by the release ofinflammatory cytokines and commensal bacteria have beenshown to provoke anti-inflammatory responses from thegut epithelia, suggesting a mechanism of action to treatthe disease [105]. Probiotics can have an inhibitory impacton the development of NAFLD by a number of mecha-nisms: competitive inhibition of pathogenic bacterial strains,alteration of the inflammatory effects of pathogenic strainsthrough changes in cytokine signalling, improvement of thefunction of the epithelial barrier and direct decreases ofproinflammatory cytokines, including TNF-α [105]. VSL#3is a high-potency medical food probiotic made up of anumber of different bacterial strains [106]. These strains

make up 450 billion live lactic acid bacteria per packet: Bifi-dobacterium breve, Bifidobacterium longum, Bifidobacteriuminfantis, Lactobacillus acidophilus, Lactobacillus plantarum,Lactobacillus paracasei, Lactobacillus bulgaricus, and Strepto-coccus thermophilus [106]. This combination, in both murineand human trials, demonstrated all of the mechanismsdescribed as potential beneficial targets for the treatmentof NAFLD [107]. Murine models of acute liver injuryhave also shown a decrease in hepatic injury following theadministration of various Lactobacillus and Bifidobacteriumspecies [108–110].

There are a number of mechanisms by which probioticscould be exerting their beneficial effects, as shown inFigure 2. The mechanisms include (1) by the productionof pathogen inhibitory substances; (2) by the blocking ofpathogenic bacteria adhesion sites; (3) by nutrient compe-tition and production; (4) by the degradation of toxins andtoxin receptors; (5) by the modulation of immune responses[104].


Poly-L-Lysine

Alginate coat

1–1000 μm

(a)

Genipincrosslinked

chitosan

Alginate corecontaining

bacterial cells

(b)

Figure 3: The concept of microcapsules for probiotic delivery. (a) Alginate-Poly-L-Lysine (APA) and (b) Genipin Crosslinked AlginateChitosan (GCAC) microcapsules.

4.2. System for Delivering Probiotics to the GIT. Probioticsmust be delivered to the target sites in sufficient numberand metabolic active phase to be effective. Currently avail-able probiotic formulations are excellent but have seriouslimitations. One of the major limitations is the delivery ofprobiotics to the lower GIT, with the presence of acids andbile greatly hindering the viability of the probiotics as theytravel through the gut (specifically the acidic environment ofthe stomach). A delivery system is, hence, required to surpassthis obstacle. Another complication is the presence of animmune system which can be induced and potentially attackthe delivered cells. Hence, a method is required to protectthe probiotic cells while maintaining high levels of probioticviability and activity when delivered in the GIT. There aremany methods available, each with its own limitations.This paper introduces microencapsulation and discusses itspotentials and limitations in bacterial cell delivery to the GIT.

5. Microencapsulation andDelivery of Probiotics

Microencapsulation is a method defined as the “entrapmentof a compound or a system inside a dispersed material for itsimmobilization, protection, controlled release, structurationand functionalization” [111]. There exists a great variety ofmicrocapsules which can differ in size, composition, andfunction, depending on the final goal of the encapsulatedproduct. Microcapsules can be used to entrap all sorts ofsubstances: solids, liquids, drugs, proteins, bacterial cells,stem cells, and so forth [112–114]. With such a range ofsubstances that can be entrapped, microcapsules can have anassortment of objectives and applications, whether for drugdelivery, enzyme retrieval, artificial cell and artificial organdelivery or, as described in this review, for the delivery of liveprobiotic bacteria.

There are a number of microcapsule delivery systems thathave been proposed for the oral delivery of live bacterialcells, as detailed in Table 1. Sun and Griffiths investigatedthe use of acid-stable beads made of gellan and xanthangum for the immobilization of Bifidobacterium [84]. Theresearch group demonstrated that immobilized cells survivedsignificantly better than free cells after refrigeration in

pasteurized yogurt for a period of 5 weeks [84]. One com-mon encapsulation method, for viable cell immobilization,utilizes calcium alginate as a polymer [115]. However, oneprominent difficulty encountered with the use of alginatebeads is that these, alone, are not acid resistant and uponexposure to the low pH conditions encountered in thestomach, display significant shrinkage and a decrease inmechanical strength [64]. A number of methods utilizingpolymer cross-linking have been suggested, including for-mulations using carrageenan, alginate-poly-L-lysine, starchpolyanhydrides, polymethacrylates, and enteric coated poly-mers [116]. Microencapsulation methods are still beingdeveloped and optimized to allow for increased gastrointesti-nal survival and immunoprotection. One newly developedtype of microcapsule that shows promising results in termsof mechanical stability and pH resistance is the genipin-crosslinked-alginate-chitosan (GCAC) microcapsule, shownin Figure 3 [87, 117].

One of the most commonly utilized and characterizedformulations for microencapsulation is the alginate-poly-L-lysine-alginate (APA) microcapsule [118]. This type ofmicrocapsule has been used for many applications includingdrug, stem cell, and bacterial cell delivery. This method relieson a polyelectrolyte complexation mechanism for the asso-ciation of the polymers, alginate and poly-L-lysine (PLL).Alginate is a naturally occurring biocompatible polymer,extracted from brown algae, that is increasingly being used inthe biotechnology industry for a wide range of applications[119]. Alginate is an unbranched polysaccharide which con-tains 1,4′-linked β-D-mannuronic acid and α-L-guluronicacid blocks which are interdispersed with regions of thealternating structure, β-L-mannuronic acid-α-L-guluronicacid blocks [120]. PLL is a polypeptide made up of theamino acid L-lysine that is available in a variable numberof chain lengths, determined by its molecular weight. It isa polycationic polymer that can be used during the coatingstep of microencapsulation. The addition of this polymerleads to the formation of a capsule membrane that providesselective permeability and immunoprotection. The alginatebead could not withstand the harsh conditions of the GITin the absence of PLL, which provides it with an increasedmechanical stability.


Table 1: Types of microcapsules available for the targeted deliveryof probiotic bacteria.

Types of Microcapsules Bacteria Reference(s)

Alginate Beads

L. rhamnosusB. longumL. salivariusL. plantarumL. acidophilusL. paracaseiL. caseiB. lactisL. reuteri

[64–70]

Alginate-celluloseacetate phthalate

B. lactisL. acidophilus

[71]

Alginate-chitosanB. animalis subsp. lactisL. bulgaricus

[72, 73]

Alginate-chitosan-Acryl-Eze

B. animalis subsp. lactis [72]

Alginate-chitosan-alginate

B. bifidumL. casei

[64]

Alginate-chitosan-Sureteric

B. animalis subsp. lactis [72]

Alginate-coated gelatinB. adolescentisB. pseudolongum

[74]

Alginate-poly-L-lysine-alginate

B. bifidumL. reuteriL. casei

[64, 75]

Alginate-starch

L. acidophilusB. lactisB. infantisL. casei

[76–78]

Gelatin-gumarabic-soluble starch

B. infantisB. longum

[79–81]

Gelatin-toluene-2-4-diisocyanate

L. lactis [82]

Gellan-alginate B. bifidum [83]

Gellan-xanthan

B. adolescentisB. bifidumB. breveB. infantisB. lactisB. longum

[84–86]

Genipin-crosslinked-alginate-chitosan

L. plantarum [87]

Pectin-caseinB. lactisL. acidophilus

[88]

Potato starchgranules-amylose

B. longum [89]

Whey proteinB. breveB. longumL. rhamnosus

[90, 91]

κ-carageenan

B. longumS. thermophilusL. bulgaricusS. lactis

[92, 93]

There are a number of different methods used to fabricatemicrocapsules. The microencapsulation technique employedis determined by the type and the size of microcapsules

one wants to obtain. The characteristics of the microcapsulemust also take into consideration the function that themicrocapsule will ultimately undertake. There are generallythree main stages to the process of microencapsulation.The first step is the incorporation of the ingredients intoa solution by mixing or dispersion, to make up the coreof the microcapsule. This is then followed by mechanicaloperations, such as spraying or emulsification, to formthe droplets. The final step of microencapsulation involvesproduct stabilization through coating, followed by a num-ber of physical or chemical processes [111]. Each stepof microencapsulation can be optimized according to thedesired characteristics of the final formulation.

6. Microencapsulated Probiotics

There has been strong interest in the field of microencapsu-lated probiotics. Research has shown that microencapsulatedprobiotics keep their viability better than free cells understress in GIT deliveries. Research into the applications ofmicroencapsulated probiotics is also ongoing, with promis-ing results for the eventual treatment of a number ofdisorders, described in the following section.

6.1. Microencapsulated Probiotics and Colon Cancer. Thepotential antitumorigenic properties of a microencapsulatedformulation of L. acidophilus were studied in Min (multipleintestinal neoplasia) mice carrying a germline Apc mutationwhich spontaneously develop numerous pretumoric intesti-nal neoplasms [29]. The mice were gavaged APA microcap-sules of L. acidophilus over a period of 12 weeks followed bythe enumeration, the classification and the histopathologyof adenomas [29]. Unfortunately, no statistically significantdifference was observed between the treatment and controlgroup in terms of the number of large intestinal (colonic)adenomas [29]. On a more positive note, there was a sta-tistical difference between the control and treatment groupsfollowing analysis of the small intestine number of adenomasand gastrointestinal intraepithelial neoplasias [29]. Thesepreliminary results suggest that microencapsulated probioticbacteria could have a role in the development of a successfulcolon cancer therapeutic.

6.2. Microencapsulated Probiotics for Use in CardiovascularDiseases. Recently, microcapsules containing bacterial cellshave been developed as a cholesterol lowering therapy. Earlyresearch has demonstrated that certain Lactobacilli have abile salt hydrolase (BSH) enzyme which can contribute toa significant cholesterol lowering effect in vivo in cardio-vascular diseases [121]. This enzyme contributes to thedeconjugation of bile salts in the intestine [121]. The oraldelivery of Lactobacillus has, therefore, emerged as a potentialmechanism for inducing cholesterol lowering. Martoni et al.demonstrated that microencapsulated BSH-active bacteriaare able to survive in a simulated human gastrointestinalmodel while maintaining cell viability and enzyme activity,which would not be possible with the direct delivery ofnonmicroencapsulated bacterial cells [75].


Another microencapsulated probiotic Lactobacillus dem-onstrated cholesterol lowering capabilities in hypercholes-terolemic animals, albeit with a different mechanism ofaction involving a feruloyl esterase enzyme [122]. Lacto-bacillus fermentum, a feruloyl esterase active bacterium, wasmicroencapsulated and delivered to hypercholesterolemichamsters twice daily by oral gavage, for a period of 18 weeks[122]. Following treatment, hamster serum cholesterol, LDLcholesterol, and the atherogenic index were 21.36%, 31.40%,and 32.59% lower, respectively, in the treatment group whencompared to the control group [122]. Histological studieswere also performed and demonstrated that the microencap-sulated probiotic reduced the progression of atheroscleroticlesions in the test animals [122]. This probiotic formulationwas hence shown to be effective at managing excessive serumcholesterol and triglyceride levels [122]. With these results,the microencapsulation of probiotics is very promising forthe development of a cholesterol-lowering therapeutic incardiovascular diseases.

Microencapsulation has the potential to be useful inother disease applications. It has been shown that, to beeffective at reducing colon tumorigenesis, therapeutic pro-biotic microorganisms must remain viable in vivo [123]. Thesame study that demonstrated that the administration of L.acidophilus had an inhibitory effect on colon tumorigenesisshowed that the amount of probiotic colonization of the GITis directly linked to the rate of inhibition of tumorigenesis[98]. Since viability is vital to the mechanism of action ofthe probiotic, it is crucial to realize that, of the bacteriaingested, only 1% survive the gastric transit, limiting theoverall therapeutic effect of any orally delivered bacterialformulation [124]. With unprotected probiotic formulationsalready demonstrating therapeutic potential, microencapsu-lation could prove beneficial in increasing efficacy.

7. Challenges and Future Outlooks

The gut microbiota is a complex system that has been shownto influence health. Although probiotics, prebiotics, andsynbiotics have shown great potential for the treatment ofa number of disorders, there are still a number of challengesthat remain to be addressed before they can be successfullyused to treat/prevent disorders. Microencapsulation hasprovided a significant advancement in the field, allowingfor the delivery of a greater number of viable bacteriato the GIT. However, a number of issues concerning theformulation of a microencapsulated probiotic still need to beaddressed before a successful product can be developed. Theprocess and methods for microencapsulation require furtherinvestigations and optimization. As aforementioned, thereare a number of microencapsulation types being employed,each varying in efficiency and application. Furthermore, theindustry scale production of microencapsulated probiotics,at a cost-effective level, and investigation of formulationstability, cell viability and retention of metabolic activity ofthe encapsulated bacterial cells requires further developmentfor specific bacterial strains and diseases.

Most of the probiotics are strain specific, they thereforemust be developed and characterised in vitro and evaluated

for their suitability and efficacy in proper animal models andhuman clinical trials. Furthermore, a specific mechanism ofaction must be developed for each application so that anevidence-based probiotic formulation can be designed thatcan potentially compete with well-articulated and well-developed drug formulations. The elucidation of the mech-anism of action of the probiotic would allow for a betterselection process. As described before, the administrationof bacteria from the same species but of different strainsresulted in noncomparable effects, further emphasizing theimportance of mechanistic studies as part of the probioticselection process. The variability in experimental designposes a great challenge in probiotic research that must beaddressed properly. Since the compositions of the gut micro-flora are not identical, there can be contradicting resultsas to the beneficial effect of probiotics on the microbiotaof the GIT. This composition variability also gives rise topotential difficulties in terms of the use of animal models forthe investigation of probiotic formulations. A targeted well-defined formulation should be developed in which micro-encapsulation will play a critical role.

There are excellent trials available that demonstrate theefficacy of these formulations but safety is an issue thatremains to be investigated. As mentioned earlier, rigorousin vitro and in vivo animal and human clinical studies areneeded to demonstrate the efficacy and the long-term safetyof microencapsulated and other probiotic formulations.Nevertheless, the literature suggests that probiotics willlead to efficient therapeutic formulations for the treatmentand/or prevention of a number of animal and human healthdisorders.

Acknowledgments

The authors would like to acknowledge the CanadianInstitute of Health Research (CIHR) Grant (MPO 64308)and grants from Micropharma to Dr. S. Prakash, the sup-port of the Industrial Innovation Scholarship (IIS) BMPInnovation—NSERC, FQRNT and Micropharma Limited toC. T. Duchesneau, and the NSERC Undergraduate StudentResearch Award to Arielle Cantor.

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